JP2000201675A - Heat-resistant creatine amidinohydrolase and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new heat-resistant enzyme having such catalytic activity as to hydrolyze creatine to form sarcosine and urea, therefore to be used for determining the creatine levels in human body fluids and useful as e.g. a diagnostic for various diseases including nephropathy. SOLUTION: This enzyme, a new heat-resistant creatine amidinohydrolase, has the following physicochemical characteristics: having such a catalytic activity as to hydrolyze 1 mol creatine to form 1 mol sarcosine and 1 mol urea; having substrate specificity to creatine, an optimum pH of 7.0-8.0, a stable pH range of 4.0-11.0, and a suitable action temperature of about 45 deg.C; stable at 53 deg.C; having a molecular weight of 92,000 (gel filtration method). Being capable of determining the creatine levels in human serum or urine, this enzyme is useful as e.g. a diagnostic for various diseases including nephropathy. This new enzyme is obtained by integrating Escherichia coli with a recombinant plasmid containing Alcaligenes sp. KS-85 strain-derived creatine amidinohydrolase gene which is then expressed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a thermostable creatine amidinohydrolase and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION Creatine amidinohydrolase is
An enzyme that has the catalytic activity of hydrolyzing creatine to produce sarcosine and urea, can be used to measure the amount of creatine in human serum or urine, and is a diagnostic agent for various diseases including kidney disease Can be used as Conventionally, creatine amidinohydrolase derived from the genus Alcaligenes has a Km value of about 13 mM and a heat resistance of about 45 ° C., so that a lower K
A creatine amidinohydrolase that exhibits an m value and that is stable against higher temperatures has been demanded in consideration of the future distribution and sale of reagents in liquefaction. Furthermore, conventionally, when the heat resistance of creatine amidinohydrolase was performed by genetic modification, the Km value was the same as that of the original strain,
In many cases, the heat resistance and the Km value were improved by modification.

[0003]

An object of the present invention is to provide a thermostable creatine amidinohydrolase and a method for producing the same.

[0004]

Means for Solving the Problems The present inventors have further studied in view of the above-mentioned object, and as a result of genetically modifying the creatine amidinohydrolase gene derived from alkaligenes (described in JP-A-8-89255), The present inventors have found that sex creatine amidinohydrolase can be obtained, and have completed the present invention.

That is, the present invention is a thermostable creatine amidinohydrolase having the following physicochemical properties: (a) action: hydrolyzing one mole of creatine to produce one mole of sarcosine and one mole of urea; I do. (B) substrate specificity; creatine has substrate specificity. (C) optimum pH; 7.0 to 8.0 (d) stable pH range; 4.0 to 11.0 (e) range of suitable temperature for operation; around 45 ° C (f) thermal stability; 53 ° C (g ) Molecular weight: 92,000 (gel filtration method) In addition, the present invention comprises culturing a microorganism having the above-mentioned physicochemical properties and capable of producing heat-resistant creatine amidinohydrolase, and converting heat-resistant creatine amidinohydrolase from the culture. A method for producing thermostable creatine amidinohydrolase, which comprises collecting.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The thermostable creatine amidinohydrolase of the present invention,
For example, it can be obtained as follows. First, the isolated alkaligenes sp. Recombinant plasmid pUCE100 DNA containing the creatine amidinohydrolase gene derived from the KS-85 strain (JP-A-8-8925)
No. 5) described in E. coli JM109.
(PUCE100) (FERM BP-4803) is extracted and purified by using, for example, QIAGEN (manufactured by Funakoshi). The vector DNA that can be used in the present invention is not limited to the above-described plasmid vector DNA, but may be other ones such as bacteriophage vector DNA and plasmid beta DNA. Specifically, p
UC118 (manufactured by Takara Shuzo) or the like is preferable.

[0007] Next, one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and the creatine amidinohydrolase activity, preferably, the heat-resistant creatine amidinohydrolase activity Any method may be used to obtain a thermostable creatine amidinohydrolase gene encoding a protein having, for example, a recombinant mutagenized DNA, for example, using a chemical mutagen such as hydroxylamine or nitrite or a PCR method. Point mutation such as random conversion, site-specific mutagenesis, which is a well-known technique for generating site-specific substitution or deletion mutation using a commercially available kit; selective cleavage of this recombinant plasmid DNA And then the selected oligonucleotides De removing or adding a method for coupling; oligonucleotide mutagenesis method.

[0008] The recombinant DNA after the above treatment is purified using a desalting column, QIAGEN (manufactured by Funakoshi) and the like.
Various recombinant DNAs are obtained.

The various recombinant DNAs thus obtained are used to transform or transform E. coli K12, preferably E. coli JM109 (manufactured by Toyobo), XL1-Blue (manufactured by Funakoshi Co., Ltd.), etc. After the introduction, a transformant or a transductant containing a recombinant DNA having various creatine amidinohydrolase gene fragments can be obtained.

[0010] For example, in the case of a transformant, desired properties are obtained from the obtained transformant (which contains recombinant plasmid DNA containing various thermostable creatine amidinohydrolase genes). The following method can be used to obtain a strain producing thermostable creatine amidinohydrolase having (low heat resistance and low Km value).

First, the above-mentioned transformant was used for each colony in a TY medium (50 μg Ampicillin, lm).
(M IPTG addition) and the like to induce various heat-resistant creatine amidinohydrolases contained in the recombinant plasmid DNA. After the culture, the obtained culture is subjected to ultrasonic crushing, and the crude enzyme extract is subjected to measurement of the residual activity after heat treatment at 50 ° C. for 30 minutes and the Km value based on a line weber-Burk plot. In addition, the target transformant is selected by comparing the measurement results of each mutant strain with the extracted, treated, and measured wild-type protein in the same manner.

A heat-resistant creatine amidinohydrolase-producing transformant or transductant obtained as described above, preferably a strain belonging to the genus Escherichia, is used to produce heat-resistant creatine amidinohydrolase. Can be performed as follows. In order to culture the above microorganism, it may be cultured by a usual solid culture method, but it is preferable to employ a liquid culture method as much as possible.

Examples of the medium for culturing the microorganisms include yeast extract, peptone, meat extract, corn steep liquor, leachate of soybean or wheat koji, and the like.
More than one nitrogen source includes potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ferric chloride, and sulfuric acid
One or more inorganic salts such as iron or manganese sulfate are added, and if necessary, saccharide raw materials, vitamins and the like are appropriately added.

The initial pH of the medium is suitably adjusted to 7-9. The cultivation is preferably carried out at 30 to 42 ° C, preferably around 37 ° C for 6 to 24 hours, by aeration and stirring deep culture, shaking culture, static culture and the like. After completion of the culture, a normal enzyme collecting means can be used to collect the thermostable creatine amidinohydrolase from the culture.

The cells are separated from the culture by, for example, filtration, centrifugation, etc., and washed. It is preferable to collect thermostable creatine amidinohydrolase from the cells. In this case, the cells can be used as they are, but a method of destroying the cells using various disrupting means such as an ultrasonic crusher, a French press, and a dynamill, a cell cell wall using a cell wall lysing enzyme such as lysozyme. It is preferable to collect the thermostable creatine amidinohydrolase from the cells by a method of dissolving the enzyme, a method of extracting the enzyme from the cells using a surfactant such as Triton X-100, or the like.

To isolate thermostable creatine amidinohydrolase from the thus obtained crude enzyme solution,
A method used for ordinary enzyme purification can be used. For example, it is preferable to carry out an appropriate combination of ammonium sulfate salting out, organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, adsorption chromatography, electrophoresis and the like.

The physicochemical properties of the resulting thermostable creatine amidinohydrolase are as follows. (1) Action One mole of creatine is hydrolyzed to produce one mole of sarcosine and one mole of urea.

(2) Substrate Specificity Creatine has substrate specificity.

(3) 50 mM sodium acetate-acetic acid buffer (pH 4.0-5.5), 50 mM MES buffer (p
H5.5-6.5), 50 mM phosphate buffer (pH 6.5).
5-8.0), 50 mM Tris-HCl buffer (p
H8.0-9.0), 50 mM CHES buffer (pH
9.0-10.0) and 50 mM CAPS buffer (p
H10.0 to 11.0) at each pH
As a result of performing the enzyme reaction at a temperature of 37 ° C. for 10 minutes, the relative activities were as shown in FIG. From FIG. 1, the optimum pH of the present enzyme was 7.0 to 8.0. (4) Range of suitable temperature for action The activity of the present enzyme was measured at various temperatures using a reaction solution having the same composition as the reaction solution in the activity measurement method described later. As a result, the result was as shown in FIG. 2. As shown in FIG. 2, the range of suitable temperature for the action of this enzyme was around 45 ° C.

(5) Stable pH range As a buffer, 50 mM sodium acetate-acetic acid buffer (pH 4.0 to 5.5), 50 mM MES buffer (p
H5.5-6.5), 50 mM phosphate buffer (pH 6.5).
5-8.0), 50 mM Tris-HCl buffer (p
H8.0-9.0), 50 mM CHES buffer (pH
9.0-10.0) and 50 mM CAPS buffer (p
H10.0-11.0) and pH 4.0-11.0.
After treatment at 25 ° C. for 17 hours, the residual activity of the present enzyme was measured, and the results were as shown in FIG. FIG.
Thus, the stable pH range was pH 4.0 to 11.0. (6) Thermostability The results of thermostability when treated with 50 mM Tris-HCl buffer (pH 7.5) at each temperature for 30 minutes are as shown in FIG. It was stable up to around ° C.

(7) Enzyme activity measurement method The activity of the present enzyme was measured under the following conditions. In addition,
Enzyme activity that produces 1 micromole of urea per minute
Unit. (Reagent preparation) 1 solution; substrate solution) 6.63 g of creatine is dissolved in 500 ml of 50 mM phosphate buffer (pH 7.7). 2 liquid; coloring solution) ρ-dimethylaminobenzaldehyde 1
0 g is dissolved in 500 ml of special grade ethanol and mixed with a mixture of 575 ml of resin water and 75 ml of concentrated hydrochloric acid.

(Measurement procedure) 1) 0.9 ml of 1 solution is preincubated at 37 ° C. for 5 minutes. 2) 0.1 ml of an enzyme solution (adjusted to about 1 to 2 U / ml) is mixed and reacted at 37 ° C. for 10 minutes. 3) After the reaction for 10 minutes, 2 ml of the above two liquids are mixed. 4) After mixing with the two liquids, the mixture was left at 25 ° C. for 20 minutes.
Measure the absorbance at 35 nm. (ODsample) 5) The blank value was measured by incubating 0.9 ml of 1 solution at 37 ° C. for 10 minutes, mixing 2 ml of the above 2 solutions, further mixing 0.1 ml of the enzyme solution, and adding 20 ml at 25 ° C.
This was performed by allowing the mixture to stand for a minute and then measuring the absorbance at 435 nm. (ODblank)

(Calculation of activity) U / ml = ΔOD * × 18.06 ** × dilution ratio * (ΔOD = ODsample−ODblank) (* coefficient calculated from urea calibration curve)

(8) Km value The Km value of the present enzyme was measured using the above-mentioned activity measurement method.
The value was 8.6 mM (relative to creatine). (9) Molecular weight 92,000 (gel filtration method)

[0025]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

(Example 1) (1) Preparation of recombinant plasmid pUCE100 DNA E. coli JM109 (pUCE100)
(FERM BP-4803) in TY medium (1% Bacto Tryptone, 0.5% Peptone, 0.25% NaC)
l) Inoculated into 20 ml and shake-cultured at 37 ° C for 18 hours to obtain a culture. The culture was collected by centrifugation at 6000 rpm for 10 minutes to obtain cells. The recombinant plasmid pUCE100 DNA was extracted from the cells using QIAGEN tip-100 and purified.
70 μg of the recombinant plasmid pUCE100 DNA
Obtained.

(2) Mutation operation Of the recombinant plasmid DNA (100 μg), 2 μm
Using XL-RED (manufactured by STRATAGENE) (prone to cause an error in replication of plasmid and cause mutation during propagation) using D. g. M. The method of Morrison (Methodin Enzymology, 68,
326-331, 1979).
500 transformants obtained from 500 transformants were all inoculated into 20 ml of TY medium and cultured with shaking at 37 ° C. for 18 hours. After culturing, the culture is grown at 6000 rpm for 1 hour.
The cells were collected by centrifugation for 0 minutes to obtain cells. The plasmid pUCE100 was extracted and purified from the cells using QIAGEN tip-100 (manufactured by Funakoshi), and the recombinant plasmid pUCE100 DNA to be mutated was extracted.
Was obtained in an amount of 70 μg. Using 5 μg of this plasmid,
M. Morrison's Method (Method in E
nzymology, 68, 326-331, 197
As a result of transforming Escherichia coli JM109 strain (manufactured by Toyobo Co., Ltd.) according to 9), about 2,000 transformed strains having about 2,000 mutated plasmids were obtained.

(3) Screening of mutant strains having improved heat resistance and reactivity with a substrate First, 2 ml of the obtained transformant was used for each colony.
TY medium (50 μg Ampicillin, 1 mM I
(PTG addition), and various heat-resistant creatine amidinohydrolases contained in the plasmid were induced and produced. After culturing, the obtained culture was subjected to ultrasonic crushing, and the residual activity of the crude enzyme extract after heat treatment at 50 ° C. for 30 minutes was measured.・ K by Bark's plot
The m value was measured. In addition, the measurement results of each mutant were subjected to extraction treatment in the same manner, and compared with the measured wild-type creatine amidinohydrolase, and a mutant satisfying the purpose was selected, and the mutant Escherichia coli (E. coli) was selected.
Thermostable creatine amidinohydrolase was obtained from JM109 (pUCE100 B-40). E. coli JM109 (pUCE100 B-4)
0) is a FERM to the Institute of Biotechnology and Industrial Technology
Deposited as P-15971.

Example 2 A mutant E. coli JM109 (pUCE10) obtained as described above was used.
0 B-40) in TY medium containing 1 mM isopropyl-β-D-galactoside (1% bacto-tryptone,
0.5% Bacto-yeast extract, 0.5% N
aCl, pH 7.5) After shaking culture in a 100 ml dispensed Sakaguchi flask for 16 hours, 3
A flame was inoculated into 20 L of a TY medium in a 0 L culture tank. After inoculation, the number of rotations was 450 rpm, the air flow was 20 L / min,
C. for about 20 hours.

After completion of the culture, the cells were collected from 20 L of the culture solution using a Microser (PW-303, manufactured by Asahi Kasei Corporation), and washed with a 20 mM phosphate buffer (pH 7.5). The cells were suspended in about 10 L of the same buffer. Step 1 (adjustment of crude enzyme solution):
L), lysozyme 20 g (50 mM phosphate buffer, pH
7.5, 100 ml) and 0.55 M EDTA, pH
After adding and mixing 8.0 L and leaving the mixture at 30 ° C. overnight, 500 ml of a 5% aqueous protamine solution (pH 8.0) was added dropwise with stirring to remove nucleic acid. This aqueous solution was added to a 10 mM CAPS-NaOH buffer solution (pH 10.
0) (hereinafter referred to as buffer A).

Step 2 (DEAE-cellulose treatment): Approximately 3 kg of cellulose by wet weight is added to a dialysate (about 28 L) and mixed to adsorb thermostable creatine amidinohydrolase, and then 5% glycerin and 0.
DE in buffer A containing 05% 2-mercaptoethanol
Wash AE-cellulose, then add 0.5M KCl
The heat-resistant creatine amidinohydrolase was eluted with the contained buffer solution A and ultra-concentrated. Step 3 (DEAE-Sepharose CL-4B treatment): A concentrated solution (about 1.0 L) was added with about 1.0 kg of DEAE-Sepharose CL wet weight buffered with buffer A.
-4B was added and mixed, and the DEAE-Sepharose CL-4B was washed with a buffer A containing 0.05 M KCl and adsorbing thermostable creatine amidinohydrolase,
Next, the heat-resistant creatine amidinohydrolase was eluted with buffer A containing 0.1 M KCl, and ultra-concentrated. Step 4 (Sephataryl S-200 treatment): Sephacryl S-20 in which a concentrated solution (about 1.0 L) was packed in a column
At 0, molecular sieving was performed and the active fraction (2.2 g) was collected. The specific activity of the active fraction was 9 U / OD 280 nm.

According to the present invention, thermostable creatine amidinohydrolase can be efficiently produced, and the present invention is industrially useful.

[0032]

[Sequence List] SEQUENCE LISTING <110> KIKOMAN CORPORATION <120> THERMOSTABLE CREATING
E AMIDINOHYDROLASE AND PR
OCESS FOR PRODUCING THE S
AME <130> P2035 <160> 1 <210> 1 <211> 404 <212> PRT <213> Alcaligenes sp. <400> 1

[Brief description of the drawings]

FIG. 1 is a diagram showing the optimum pH of the present enzyme.

FIG. 2 is a graph showing a suitable temperature range for the action of the present enzyme.

FIG. 3 is a view showing a stable pH range of the present enzyme.

FIG. 4 shows the thermostability of the present enzyme.

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[Procedure amendment]

[Submission date] September 7, 1999 (1999.9.7)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction contents]

(3) Screening of mutant strains having improved heat resistance and reactivity with a substrate First, the obtained transformant was transformed with 2 ml of T
Liquid culture was performed in a Y medium (50 μg Ampicilin, supplemented with 1 mM IPTG) to induce various heat-resistant creatine amidinohydrolases contained in the plasmid. After the culture, the obtained culture was subjected to ultrasonic crushing, and the residual activity of the crude enzyme extract after heat treatment at 50 ° C. for 30 minutes was measured.・
Km values were measured by Bark's plot. Also,
For the measurement results of each mutant strain, the same extraction treatment was performed, and a mutant strain satisfying the purpose was selected by comparing with the measured wild-type creatine amidinohydrolase, and the mutant E. coli (E. coli) JM109 (pUCE100 B -40), a thermostable creatine amidinohydrolase was obtained. E. coli
JM109 (pUCE100 B-40) is transferred to FERM BP-6867 (Biotech) from FERM P-15971.

[Document name] Notification of change of accession number

[Submission date] September 10, 1999 (19
99.9.10.)

[Name of former depositary institution] Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry

[Old Accession Number] FERM P-15971

[Name of the new deposited organization] Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry

[New accession number] FERM BP-6867

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:19) F-term (Reference) 4B050 CC01 CC03 DD02 LL03 4B065 AA26X AA26Y AC02 BA02 BA16 CA31 CA46

Claims (2)

[Claims] 1. A thermostable creatine amidinohydrolase having the following physicochemical properties. (A) action; hydrolyze 1 mole of creatine to produce 1 mole of sarcosine and 1 mole of urea. (B) substrate specificity; creatine has substrate specificity. (C) optimum pH; 7.0 to 8.0 (d) stable pH range; 4.0 to 11.0 (e) range of suitable temperature for operation; around 45 ° C (f) thermal stability; 53 ° C (g ) Molecular weight: 92,000 (gel filtration method) 2. A heat-resistant creatine amidinohydrolase having physicochemical properties according to claim 1, wherein the microorganism is capable of producing heat-resistant creatine amidinohydrolase, and the heat-resistant creatine amidinohydrolase is collected from the culture. A method for producing creatine amidinohydrolase.